CN102533845B - Agrobacterium tumefaciens-medicated genetic transformation method of wheat - Google Patents
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Abstract
The invention discloses an agrobacterium tumefaciens-medicated genetic transformation method of wheat. The agrobacterium tumefaciens-medicated genetic transformation method comprises the following steps of: firstly, inoculating recombinant Agrobacterium tumefaciens containing a target DNA (Deoxyribonucleic Acid) into wheat immature embryos; and then co-culturing the inoculated wheat immature embryos for enabling the T-DNA of the recombinant Agrobacterium tumefaciens to be integrated with a chromosome of the wheat; and secondly, directly inoculating the wheat immature embryos co-cultured in the first step in a long-seedling culture medium for culturing to obtain transgenic wheat seedlings with roots, stems and leaves. The method disclosed by the invention has the advantages that the applicability is wide, so that the method is suitable for transforming various wheat species; the method is simple in operation, and thus a transgenic plant can be obtained without inducing a tissue culture and regenerating the plant; and the transformation period is short, i.e. the process from the infection to the obtaining of the transgenic wheat seedlings with the roots, the stems and the leaves only requires 11-14 days. The method has very important significance for genetic improvement on the wheat species and the obtaining of new species of the transgenic wheat.
Description
Technical field
The present invention relates to a kind of agriculture bacillus mediated Efficiency of Wheat Transformation method.
Background technology
Wheat is one of important cereal crop of depending on for existence of the mankind, and worldwide cultivated area and ultimate production have all exceeded other farm crop, and its genetic improvement is the important goal that people make great efforts all the time.Traditional wheat breeding technology exists the limitation such as breeding cycle is partially long, yield potential is limited, along with the development of biotechnology, utilizes genetic engineering technique to carry out breed improvement to wheat and more and more comes into one's own.Aspect the research of plant gene conversion system, wheat as paddy rice, corn etc., shows hysteresis quality and difficulty with respect to other cereal crop.Efficiency of Wheat Transformation research starts from phase late 1980s, because wheat is not the natural host of Agrobacterium, and wheat protoplast regeneration is relatively difficult again, so until the people such as Vasil in 1992 successfully import wheat callus by GUS and Bar gene by particle bombardment, and after obtaining transfer-gen plant, about the report of wheat transgenic just starts to increase.At present, transgenic paddy rice has entered interim test and safety evaluation stage, and the research of wheat transgenic obviously falls behind, the also foundation in transformation system and improving the stage.
In plant genetic engineering, utilize agriculture bacillus mediated transgenosis be at present research at most, transformation mechanism is the clearest, technological method is the most ripe gene transformation approach.Compared with via Particle Bombardment Transformation method, Agrobacterium-mediated Transformation method has a lot of advantages, and as imported large fragment goal gene, and goal gene mostly is single copy or low copy; Easily get rid of the plasmid dna sequence of redundancy; Insert the border sequence that border mostly is T-DNA, rearrangement rate is low etc.This method can be become to the focus of studying before and after the nineties for monocotyledonous transgenosis such as wheats.At that time, a kind of tendentious viewpoint was to think that monocotyledons is not the natural host of Agrobacterium, utilized agrobacterium mediation converted monocotyledons may or there is no hardly possibility.The representative figure of this viewpoint is the Switzerland scientist Potrykus that once had achievement at Zhuo aspect the cereal crop tissue culture such as wheat and via Particle Bombardment Transformation, and he has delivered his pessimistic point of view on internal authority magazine Bio/Technology and Nature.Along with the acquisition of agriculture bacillus mediated officinalis transfer-gen plant, the particularly acquisition of agriculture bacillus mediated transgenic paddy rice and transgenic corn plant, not only having changed monocotyledons is not the viewpoint of Agrobacterium natural host, and proves that agrobacterium-mediated transformation completely can be for the genetic transformation of gramineous crop.But the Agrobacterium-mediated Transformation of wheat remains a difficult problem in the world up to now.In recent years, the research group of lot of domestic and international has carried out a large amount of research and exploration from multi-angles such as wheat genotypes, agrobacterium strains, common culture condition, explant type, proposed a lot of cover genetic conversion systems, but all without any a set of, can be widely used.
At present, for the wheat explant of agrobacterium mediation converted, mostly be the embryo callus that rataria and mature embryo form, they all need to cultivate through in vitro tissue the transfer-gen plant of regenerating.But the different genotype of wheat has very large impact for the regeneration of plant, this has further limited by tissue culture approach and has obtained transgenic wheat.
Summary of the invention
The object of this invention is to provide a kind of agriculture bacillus mediated Efficiency of Wheat Transformation method, the step that the method forms without callus, has advantages of wide adaptability, simple to operate, the transformation period is short.
Method provided by the present invention is, by agriculture bacillus mediated, target DNA is proceeded to wheat cell, comprises the steps:
1) the restructuring Agrobacterium that contains target DNA is inoculated in to wheat immature embryo, then postvaccinal described wheat immature embryo is carried out to common cultivation and make the described restructuring T-DNA of Agrobacterium and the chromosomal integration of described wheat;
2) by through step 1) the described wheat immature embryo cultivated altogether is directly inoculated in long seedling substratum and cultivates, and obtains having root, the seedling of stem and leaf.
In aforesaid method, dead in order to prevent wheat immature embryo easy brownization after Agrobacterium is infected, in step 1) described the restructuring Agrobacterium that contains target DNA is inoculated in to wheat immature embryo before, described wheat immature embryo also passes through 12-24 hour, the preculture as 24 hours;
Described preculture specifically can be carried out according to the method comprising the steps: by preculture substratum dark cultivation 12-24 hour at 25 ℃ for described wheat immature embryo, as 24 hours; The basic medium of described preculture substratum can be MS substratum.
In aforesaid method, described Agrobacterium can be Agrobacterium rhizogenes, as Agrobacterium rhizogenes R1000.
In aforesaid method, described wheat immature embryo is the described wheat immature embryo of 13-15 days after blooming.
In aforesaid method, step 1) described the restructuring Agrobacterium that contains target DNA is inoculated in to wheat immature embryo, according to the method comprising the steps, carry out: described wheat immature embryo is soaked in the bacterium liquid of described restructuring Agrobacterium to 5～10 minutes, as 5 minutes; The OD of described restructuring Agrobacterium bacterium liquid
600be 0.5～0.6, as 0.6.
In aforesaid method, the described bacterium liquid 20～30s that contains described wheat immature embryo by ultrasonication between described soak period, as 20s, described hyperacoustic frequency is that 27kHz, power are 80w.
In aforesaid method, step 1) described altogether cultivation specifically can carry out according to the method comprising the steps: by step 1) the common culture medium of described postvaccinal described wheat immature embryo cultivates, in order to prevent described postvaccinal described wheat immature embryo browning, in the described substratum of cultivating altogether, can add antioxidant, described antioxidant specifically can be dithiothreitol (DTT); In order to promote the described restructuring T-DNA of Agrobacterium and the chromosomal integration of described wheat, in the described substratum of cultivating altogether, also can add Syringylethanone.
In aforesaid method, step 1) the described time of cultivating is altogether 2-3 days, as 3 days.
In aforesaid method, in order to remove non-transformed cell, in step 2) can contain the selective pressure of screen transformant in described long seedling substratum, in order to suppress the growth of Agrobacterium, in step 2) also can add the microbiotic that inhibition Agrobacterium grows in described long seedling substratum.
In aforesaid method, described preculture substratum specifically can be the MS substratum containing caseinhydrolysate 1.0g/L and 2,4-D 2mg/L, pH5.8; The described substratum of cultivating altogether specifically can be the MS substratum containing Syringylethanone 150 μ mol/L and dithiothreitol (DTT) 1g/L, pH5.2; Described long seedling substratum specifically can be the MS substratum containing cephamycin 200mg/L and Totomycin 100mg/L, pH5.8.
In method of the present invention, do not comprise the step that forms callus.
Method of the present invention can be applicable to all wheats, is particularly suitable for common wheat.
Method of the present invention has the following advantages: wide adaptability, is suitable for the conversion of multiple wheat breeds; Simple to operate, without the step of callus induction and plant regeneration, can obtain transfer-gen plant; Transformation period is short, from infecting the transgenic wheat seedling that has a root, stem and leaf to acquisition, only needs 11-14 days.The present invention is of great significance for the genetic improvement of wheat breed, the new germ plasm tool of acquisition transgenic wheat.
Accompanying drawing explanation
Fig. 1 is T
0pCR for Transgenic plant of wheat detects.Wherein, 1 is molecular weight standard DL2000 (being followed successively by from top to bottom 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp), and 2-4 is respectively blank, positive control, negative control, and 5-9 is respectively the T of each kind
0for transfer-gen plant east agriculture 7742-24, Hite-17, imperial wheat 9814-41, imperial wheat 26-5, imperial spoke wheat 10-32.
Fig. 2 is T
0for the Southern hybridization check of Transgenic plant of wheat.Wherein, 1-3 is respectively positive control, negative control, molecular weight standard pBR328/BamH I+Bg I+Hinf I (being followed successively by from top to bottom 4907bp, 2176bp, 1766bp, 1230bp, 1033bp and 653bp); 4-8 is respectively the T of each kind
0for transgenic positive individual plant: eastern agriculture 7742-24, Hite-17, imperial wheat 9814-41, imperial wheat 26-5 and imperial spoke wheat 10-32.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Wheat breed used in the present invention, plasmid and Agrobacterium rhizogenes are as follows in detail:
East agriculture 7742: Li Xinling, Xu Xiangling, Zhang Nannan. turn Progenies of Wheat genetic analysis and the Disease Resistance Identification of chitinase gene. Plant Physiology Communications, 2008,44 (1): 75-78, the public can obtain from Harbin Teachers' Univ..
Hite: Li Xinling, Qu Min, Xu Xiangling, Li Ji faces the research of the plasmid-mediated chitinase gene transformed wheat of .Ri. plant research, 2006,26 (3): 297-301, the public can obtain from Harbin Teachers' Univ..
Dragon wheat 9814: Li Xinling, Qu Min, Yan Yuqin, Xu Xiangling. affect the research of wheat mature embryo cultivation and plant regeneration factor. plant research, 2005,25 (1): 49-52, the public can obtain from Harbin Teachers' Univ..
Dragon wheat 26: Li Xinling, Qu Min, Yan Yuqin, Xu Xiangling. affect the research of wheat mature embryo cultivation and plant regeneration factor. plant research, 2005,25 (1): 49-52, the public can obtain from Harbin Teachers' Univ..
Dragon spoke wheat 10: Xing Quanhua, Wang Guangjin, Shi Jinfeng, Wang Yueguang, Li Zhongjie, beam Fengshan, Jin Demin, Wang Bin. β-1, the structure of 3-glucanase gene efficient expression vector and the conversion to wheat. Acta Genetica Sinica, 2003,30 (8): 717-722, the public can obtain from Harbin Teachers' Univ..
Plasmid pMLH7133-Chi: Li Xinling, Qu Min, Xu Xiangling, Li Ji faces the research of the plasmid-mediated chitinase gene transformed wheat of .Ri. plant research, 2006,26 (3): 297-301, the public can obtain from Harbin Teachers' Univ..
Agrobacterium rhizogenes R1000 (Agrobacterium rhizogenes strain R1000): Li Xinling, Qu Min, Xu Xiangling, Li Ji faces the research of the plasmid-mediated chitinase gene transformed wheat of .Ri. plant research, 2006,26 (3): 297-301, the public can obtain from Harbin Teachers' Univ..
The formula of basic medium MS substratum used in the present invention is as table 1:
Table 1.MS culture medium prescription
YEB liquid nutrient medium: extractum carnis 5g/L, yeast powder 1g/L, sucrose 5g/L, peptone 5g/L, MgSO
4494.94mg/L, pH7.2.
YEB solid medium: add agar 10g/L in above-mentioned YEB liquid nutrient medium.
One, the structure of the restructuring Agrobacterium rhizogenes that contains target DNA
Recombinant vectors for transforming agrobacterium rhizogenes is pMLH7133-Chi, and the goal gene on this carrier is chitinase gene (Chi), and the label screening gene on this carrier is hygromix phosphotransferase (HPT) gene.
Recombinant vectors pMLH7133-Chi is proceeded to Agrobacterium rhizogenes R1000 (Agrobacterium rhizogenes strain R1000) and obtain the restructuring Agrobacterium rhizogenes R1000/pMLH7133-Chi that contains chitinase gene.Concrete grammar is as follows: (1) is the mono-bacterium colony of picking Agrobacterium rhizogenes R1000 from containing the YEB solid plate of 50mg/L Streptomycin sulphate, be inoculated in the test tube that contains 10ml YEB liquid nutrient medium (containing 50mg/L Streptomycin sulphate), 28 ℃ of shaking culture are spent the night; (2) get 400 μ l bacterium liquid and be inoculated in 40ml containing in the YEB liquid nutrient medium of 50mg/L Streptomycin sulphate, cultivate 4-6h to logarithmic phase (OD
600=0.5); (3) ice bath 30min, gets 1.5ml bacterium liquid and joins in aseptic centrifuge tube, and the centrifugal 5min of 13000rpm, abandons supernatant; (4) 800 μ l 150mmol/L CaCl for precipitation
2eddy diffusion, 4 ℃, the centrifugal 5min of 3000rpm, abandons supernatant; (5) 800 μ l 20mmol/L CaCl for precipitation
2eddy diffusion, divides in the aseptic centrifuge tube that installs to 1.5ml by every pipe 200 μ l; (6) in 200 μ l Agrobacterium competent cells, add 1 μ g recombinant vectors pMLH7133-Chi, place 30min on ice; (7) freezing 2min in liquid nitrogen, is placed in 37 ℃ of water-bath 2min immediately after taking-up; (8) add 800 μ l YEB liquid nutrient mediums, 28 ℃ of shaking culture 1-2h; (9) get 200 μ l and coat on the YEB solid medium containing 50mg/L kantlex, 50mg/L Streptomycin sulphate, with sterilized water, replace DNA to establish a negative contrast simultaneously, put in 28 ℃ of incubators; (10) single bacterium colony that the above-mentioned cultivation of picking obtains, shaking culture in 28 ℃ of liquid YEB substratum, extracts plasmid DNA, carries out pcr amplification and identifies restructuring Agrobacterium rhizogenes R1000/pMLH7133-Chi, and the method for pcr amplification is identical with following step 3.
Two, the restructuring Agrobacterium rhizogenes transformed wheat that contains target DNA
Respectively take the rataria of the agriculture 7742 of wheat east, Hite, imperial wheat 9814, imperial wheat 26, imperial spoke wheat 10 as explant, the restructuring Agrobacterium rhizogenes R1000/pMLH7133-Chi that utilization contains chitinase gene (Chi) proceeds to chitinase gene in wheat cell, with the Agrobacterium rhizogenes R1000 that does not contain plasmid pMLH7133-Chi, be converted into contrast, method for transformation is as follows:
1, explant obtains and preculture: take away the wheat immature seed of spending rear 13-15 days; Under aseptic condition, first use 70% ethanol disinfection 30s, aseptic washing 3 times, then use 0.1%HgCl
2sterilization 12min, aseptic washing 5 times; Then from immature seed, separate the long rataria for 1.5mm-2.0mm, be inoculated on preculture substratum, 25 ℃, secretly cultivate 24 hours;
Preculture substratum: containing 1.0g/L caseinhydrolysate and 2mg/L 2, the MS substratum of 4-D, pH5.8.
2, infect together and cultivate: will be soaked in OD through the pre-incubated rataria of step 1
600be 0.6 the 5min in liquid that infects containing restructuring Agrobacterium rhizogenes R1000/pMLH7133-Chi, with frequency and power, be respectively during this time the ultrasonication 20s of 27kHz and 80w, be then inoculated in common culture medium, 25 ℃, secretly cultivate 3 days;
The preparation method who infects liquid is as follows: get the restructuring Agrobacterium rhizogenes R1000/pMLH7133-Chi bacterium liquid that step 1 obtains, streak inoculation is on the YEB solid medium that contains 50mg/L Streptomycin sulphate and 50mg/L Totomycin, 28 ℃ of dark cultivations 1 day, picking list bacterium colony, be inoculated in the YEB liquid nutrient medium that 5mL contains 50mg/L Streptomycin sulphate and 50mg/L Totomycin, 28 ℃, dark, 200rpm shaken overnight is cultivated, again this 5mL nutrient solution is all added to 100ml containing 150 μ mol/L Syringylethanones, enlarged culturing in the YEB liquid nutrient medium of 50mg/L Streptomycin sulphate and 50mg/L Totomycin, when Agrobacterium grows into logarithmic phase, room temperature, the centrifugal 5min of 4000rpm collects thalline, be resuspended in containing in the MS substratum of 150 μ mol/L Syringylethanones, adjust OD
600value is 0.6,
Culture medium altogether: containing the MS substratum of 150 μ mol/L Syringylethanones and 1g/L dithiothreitol (DTT), pH5.2.
3, long seedling: by the wheat immature embryo of cultivating altogether through step 2 sterile water wash 2-3 time, be inoculated in and contain on antibiotic long seedling substratum, 25 ℃, illumination every day 16 hours (intensity of illumination 4000Lux), dark 8 hours, cultivate 7-10 days, obtain the transgenic wheat seedling with root, stem and leaf;
Long seedling substratum: containing the MS substratum of 200mg/L cephamycin and 100mg/L Totomycin, pH5.8.
4, transplant strong sprout: the transgenic wheat seedling replanting that step 3 is obtained, to containing in the little alms bowl of vermiculite, waters 1/10 every day not containing MS substratum the timing moisturizing of agar, and hot-house culture obtains T
0for Transgenic plant of wheat.
The rataria of each product grow wheat is not after containing the Agrobacterium rhizogenes R1000 of plasmid pMLH7133-Chi and infecting, all dead afterwards in the screening of step 3, without seedling, obtains.The T that infects rataria number, acquisition of each product grow wheat
0for the statistics of Transgenic plant of wheat number in Table 2.
Three, the PCR of transgenic wheat detects
1, pcr template: the each T obtaining with the embodiment 1 that SDS method is extracted
0the total DNA of plant leaf (negative control) and the plasmid pMLH7133-Chi (positive control) of the wheat breed east agriculture 7742 of not carrying out Agrobacterium-mediated Transformation of extracting for total DNA of Transgenic plant of wheat blade, by SDS method.
2, PCR primer: two terminal sequences of chitinase gene: 5 '-GCA GTG TGG AAG GCA AGC AG-3 ' (sequence 1 in sequence table) and 5 '-CTG AGA GGT GAC AAG GTC AG-3 ' (sequence 2 in sequence table).
3, PCR program: PCR response procedures is: 94 ℃ of 5min, 95 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 60s, 30 circulations, then 72 ℃ of 5min.
4, result: the object clip size of pcr amplification is 1100bp; 1% agarose gel electrophoresis detects the PCR product of each sample template, obtains altogether eastern agriculture 7742 transgenic positive plant 5 strains, 6 strains of Hite positive plant, dragon wheat 9814 is 2 strains, and imperial wheat 26 is 3 strains, and imperial spoke wheat 10 is 3 strains, average conversion is 1.02%, and result is as shown in Fig. 1 and table 2.
The detailed process that above-mentioned SDS method is extracted DNA is as follows: (1) gets the about 100mg of regeneration plant spire, puts into 1.5ml centrifuge tube, liquid nitrogen or quartzite sand grind; (2) add Extraction buffer 600 μ l, mix room temperature reaction 30min; (3) 4 ℃, the centrifugal 5min of 13000rpm, gets supernatant; (4) add isopyknic phenol chloroform (phenol: chloroform=1: 1), put upside down gently 3-5min; (5) 4 ℃, the centrifugal 5min of 13000rpm, carefully draws supernatant; (6) add isopyknic chloroform, put upside down gently 3-5min; (7) 4 ℃, the centrifugal 5min of 13000rpm, water, with equal-volume Virahol or 2 times of volume dehydrated alcohol alcohol precipitations, slowly mixes, 4 ℃ put 10min more than.This step can be spent the night; (8) 4 ℃, the centrifugal 10min of 13000rpm, abandons supernatant, with 70% washing with alcohol, precipitates and drains; (9) add 30-50 μ l TE dissolution precipitation, add 37 ℃ of temperature of 1 μ l RNase A and bathe 30min; (10) get 5-10 μ l DNA electrophoresis detection.
Extraction buffer: 200mmol/L Tris-HCl, 250mmol/L NaCl, 25mmol/L EDTA, 0.5%SDS, pH7.5.
TE：10mmol/L(pH8.0)Tris-HCl，1mmol/L(pH8.0)EDTA。
Transformation efficiency=(PCR positive plant number/infect rataria number) × 100%.
Table 2.T
0for the pcr amplification result of transgenic wheat
| Genotype | The rataria number infecting | T 0For Transgenic plant of wheat number | PCR positive plant number | Transformation efficiency (%) |
| East agriculture 7742 | 494 | 159 | 5 | 1.01 |
| Hite | 408 | 153 | 6 | 1.47 |
| Dragon wheat 9814 | 251 | 95 | 2 | 0.79 |
| Dragon wheat 26 | 290 | 82 | 3 | 1.03 |
| Dragon spoke wheat 10 | 411 | 126 | 3 | 0.73 |
| Add up to | 1854 | 615 | 19 | 1.02 |
Four, the Southern of transgenic wheat hybridization
1, the preparation of probe: use digoxigenin labeled detection kit (Roche, Cat.No.11745832910) synthetic hybridization probe, method is as follows: add 1 μ g chitinase gene DNA (take plasmid pMLH7133-Chi as template, the pcr amplification product obtaining according to the method for step 3) in 0.2ml centrifuge tube, use ddH
2o supplements and makes final volume to 16 μ l, in boiling water bath, heating 10min makes DNA sex change also fast in cooled on ice, add 4 μ l Mix Dig-High Prime, mix also of short duration centrifugal, place 1h for 37 ℃, add the EDTA (pH=8.0) of 2 μ l 0.2M or 65 ℃ of heating 10min with termination reaction ,-20 ℃ frozen standby.
2, Southern hybridization: because restriction enzyme BamHI and SacI exist single endonuclease digestion site on the T-DNA of plasmid pMLH7133-Chi, and these two restriction enzyme sites are positioned at the both sides of chitinase gene (1.1kb), this experiment cuts with restriction enzyme BamHI and SacI while enzyme 19 T that step 1 obtains
0for the total DNA of Transgenic plant of wheat blade, plasmid pMLH7133-Chi (positive control), the total DNA of plant leaf (negative control) that do not carry out the wheat breed east agriculture 7742 of Agrobacterium-mediated Transformation; According to the method for Pehanorm Brooker (molecular cloning experiment guide (third edition), Science Press,, p495-499 in 2005), carry out Southern hybridization check.
3, result: as shown in Figure 2.19 T that PCR is positive
0for Transgenic plant of wheat, all occurred the hybrid belt of 1 1.1kb left and right, and negative control plant is without any hybrid belt.
Five, the chitinase activity of transgenic wheat is measured
Be mainly endochitinase with disease-resistant relevant chitinase, and in plasmid pMLH7133-Chi, the chitinase of entrained bean chitinase gene coding is also endochitinase.Adopt glucosamine method to measure respectively 8 strain T
0for the chitinase activity of Transgenic plant of wheat (transformant) blade, take the wheat plant (contrast strain) that do not carry out Agrobacterium-mediated Transformation as contrast, measuring method is as follows respectively:
1, the extraction of chitinase and processing
1. take respectively T
0for the blade 2g of Transgenic plant of wheat and contrast strain, put into the mortar of precooling, and add 6ml 0.1M sodium citrate buffer solution (pH5.0) and a little quartz sand, in ice bath, grind to form homogenate; 2. 4 ℃, the centrifugal 15min of 12000rpm, supernatant liquor is crude enzyme liquid; 3. get 1ml crude enzyme liquid, add 1ml 0.5% chitosan (pH6.0) and 2ml 0.2M sodium-acetate (pH5.2), mix.With the chitosan and the sodium-acetate mixed solution that do not contain crude enzyme liquid, do blank simultaneously; 4. 37 ℃ of water-bath 2h, 4 ℃, the centrifugal 5min of 12000rpm, gets supernatant liquor standby.
0.5% chitosan: soak 4h with 5% Glacial acetic acid in advance, when after abundant dissolving, adjust pH6.0 with NaOH.
2, circumscribed chitinase activity is measured
1. get supernatant liquor that 1ml step 1 obtains in test tube, add 1ml H
2o and 1ml methyl ethyl diketone reagent, shake up; 2. with glass sphere, seal test tube mouth, in boiling water, heat 20min, be then cooled to room temperature; 3. add 5ml dehydrated alcohol and 1ml 1% paradimethy laminobenzaldehyde (DMAB reagent), then complement to cumulative volume 10ml with dehydrated alcohol, fully mix; 4. 65 ℃ of-70 ℃ of water-bath 10min, accelerate CO
2discharge, be then cooled to room temperature, 530nm place measures solution light absorption value.Water replaces the crude enzyme liquid of each sample to do blank.
Methyl ethyl diketone reagent: 1ml heavily steams methyl ethyl diketone and is dissolved in 50ml 0.5M Na
2cO
3in solution, preparation before using.
The p-dimethylamino benzaldehyde of DMAB reagent: 0.8g is dissolved in 30ml ethanol, adds 30ml concentrated hydrochloric acid, and it is faint yellow that solution is.
3, total chitinase activity is measured
1. get supernatant liquor that 1ml step 1 obtains in test tube, add 100 μ l 1M phosphoric acid buffers (pH7.1) and 70 μ l 3% helicases, shake up; 2. 37 ℃ of water-bath 2h, adding water to cumulative volume is 2ml; 3. 65 ℃ of-70 ℃ of water-bath 10min, accelerate CO
2discharge, be cooled to room temperature, 530nm place measures solution light absorption value.Water replaces the crude enzyme liquid of each sample to do blank.
4, endochitinase vigor calculates
According to typical curve equation: Y=329.82X-3.3249, (X represents OD
530value, Y represents glucosamine amount (μ g), linear correlation coefficient r=0.9923), total chitinase activity of each sample that mensuration is obtained and the OD of circumscribed chitinase activity
530value is converted into respectively glucosamine output, then with decomposition colloid chitosan per hour, produces 1 μ g glucosamine and be designated as an enzyme activity unit (U).
According to formula: endochitinase vigor (U)=total chitinase activity (U)-circumscribed chitinase activity (U);
5, result: in Table 3.Result shows, the T of different varieties
0for the endochitinase vigor of Transgenic plant of wheat, all significantly higher than contrast strain, and be at least contrast the more than 2 times of strain, illustrate that the chitinase gene of external source is at T
0for expressing in transgenic wheat.
The endochitinase vigor of table 3. transgenic wheat
Claims (2)
1. the method for cultivating transgenic wheat, proceeds to wheat cell by agriculture bacillus mediated by target DNA, it is characterized in that, said method comprising the steps of:
1) the restructuring Agrobacterium that contains target DNA is inoculated in to wheat immature embryo, then postvaccinal described wheat immature embryo is carried out to common cultivation and make the described restructuring T-DNA of Agrobacterium and the chromosomal integration of described wheat;
2) the described wheat immature embryo of cultivating altogether through step 1) is directly inoculated in long seedling substratum and is cultivated, obtain thering is root, the transgenic wheat seedling of stem and leaf;
The described substratum of cultivating is altogether the MS substratum containing Syringylethanone 150 μ mol/L and dithiothreitol (DTT) 1g/L, pH5.2; Described long seedling substratum is the MS substratum containing cephamycin 200mg/L and Totomycin 100mg/L, pH5.8;
Before the restructuring Agrobacterium that contains target DNA being inoculated in to wheat immature embryo described in step 1), described wheat immature embryo also passes through the preculture of 12-24 hour; Described preculture substratum is for containing caseinhydrolysate 1.0g/L and 2, the MS substratum of 4-D 2mg/L;
Described Agrobacterium is Agrobacterium rhizogenes;
Described wheat immature embryo is the described wheat immature embryo of 13-15 days after blooming;
Described in step 1), the restructuring Agrobacterium that contains target DNA is inoculated in to wheat immature embryo, according to the method comprising the steps, carries out: described wheat immature embryo is soaked in the bacterium liquid of described restructuring Agrobacterium to 5～10 minutes; The OD of described restructuring Agrobacterium bacterium liquid
600be 0.5～0.6;
The described bacterium liquid that contains described wheat immature embryo by ultrasonication in described immersion 20～30 seconds;
The time of cultivating altogether described in step 1) is 2-3 days.
2. method according to claim 1, is characterized in that: described wheat is common wheat.
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| CN108997484B (en) * | 2017-06-07 | 2020-07-24 | 中国农业科学院作物科学研究所 | Application of wheat TaWox5 gene in improving wheat transformation efficiency |
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| CN1597963A (en) * | 2004-09-22 | 2005-03-23 | 哈尔滨师范大学 | Chitinase gene of pea bean and its amino-acid squence of coded product |
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| CN1597963A (en) * | 2004-09-22 | 2005-03-23 | 哈尔滨师范大学 | Chitinase gene of pea bean and its amino-acid squence of coded product |
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| Ri质粒介导的几丁质酶基因转化小麦的初步研究;李新玲等;《中国生物工程学会第三次全国会员代表大会暨学术讨论会论文摘要集》;20011231;121 * |
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| 影响小麦成熟胚培养及植株再生因素的研究;李新玲等;《植物研究》;20050131;第25卷(第1期);49-52 * |
| 李新玲等.Ri质粒介导的几丁质酶基因转化小麦的初步研究.《中国生物工程学会第三次全国会员代表大会暨学术讨论会论文摘要集》.2001,121. |
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